Identification of priming factors that determine cardiac lineage of cardiac stem cells

越来越多的证据表明，骨形态发生蛋白（BMP）是心脏的诱导，规范和发展的关键。虽然BMP信号通过可溶性BMP结合蛋白紧密调节，但它们在心脏分化过程中如何调节BMP信号仍然未知。为了鉴定在P19细胞的早期心肌细胞分化期间负责BMP信号传导的分子，进行cDNA扣除。我们发现在心肌细胞分化过程中BMP结合蛋白Crossveinless-2（Cv 2）的双峰表达; Cv 2在时间上早于心脏转录因子如Nkx2.5和Tbx 5表达，并在P19细胞中作为BMP信号传导的抑制因子。我们建立了一个P19克隆细胞系，该细胞系含有心脏α-肌球蛋白重链启动子驱动的增强型绿色荧光蛋白基因，以通过流式细胞术监测心脏分化。在分化的前2天期间用BMP 2处理通过下游靶Smadl/5/8蛋白和Idl基因的活化抑制心肌细胞分化，而用Cv 2处理相反地抑制Smadl/5/8活化和Idl表达，导致心脏细胞的产生增加。内源性Cv 2的RNA干扰介导的敲低（KD）显示增加的Smadl/5/8活化和受损的心肌细胞分化。心脏中胚层标志物的表达减少，而Idl和内胚层标志物如Sox 7、Hnf 4和E-钙粘蛋白的表达在Cv 2激酶死细胞中被诱导。通过在分化或与亲本细胞共培养的前2天期间向培养基中添加Cv 2蛋白来拯救这些表型。这些数据表明，Cv 2可能通过在心脏发生的早期阶段抑制BMP信号而指定心脏中胚层谱系。

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation ; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smadl/5/8 protein and Idl gene, whereas treatment with Cv2 conversely inhibited Smadl/5/8 activation and Idl expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smadl/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Idl and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.